Jiang Dan, Song Zaiwei, Ma Yi, Zhang Xu, Bing Hao, Xiong Xin, Hu Yang, Dong Fei, Zhao Rongsheng
Department of Pharmacy, Peking University Third Hospital, Beijing, 100191, China.
Therapeutic Drug Monitoring and Clinical Toxicology Center, Peking University, Beijing, 100191, China.
Anal Bioanal Chem. 2025 Feb;417(4):821-834. doi: 10.1007/s00216-024-05701-2. Epub 2024 Dec 19.
Bruton's tyrosine kinase inhibitors (BTKis) exhibit significant interindividual pharmacokinetics, making therapeutic drug monitoring (TDM) a promising approach for personalized therapy. However, simultaneous quantification of multiple BTKis poses technical challenges. A unified protocol for BTKis detection would be clinically desirable. Herein, we developed and validated a novel LC-MS/MS method for the simultaneous analysis of four BTKis including ibrutinib (IBR), zanubrutinib (ZAN), orelabrutinib (ORE), and acalabrutinib (ACB) and active metabolite of IBR and ACB (DIH and ACBM, respectively) in human plasma. The samples were prepared by liquid-liquid extraction using tert-butyl methyl ether. Ibrutinb-d4 (IS) was used as an internal standard. Chromatographic separation was obtained on an XBridge C18 column and connected to an LC-30AD system coupled to an API 4000 mass spectrometer. The mobile phase comprised 10 mM ammonium acetate containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The optimized multiple reaction monitoring transitions of m/z 441.4 → 138.3, 475.4 → 304.2, 472.5 → 455.5, 428.3 → 411.5, 466.1 → 372.2, 482.2 → 388.4, and 445.5 → 142.5 were selected to inspect IBR, DIH, ZAN, ORE, ACB, ACBM, and IS, respectively. The method exhibited linearity from 1 to 1000 ng/mL (r > 0.99) for all analytes, with intra-day and inter-day precision of 1.8 to 9.7% and accuracy below 15%. Recovery ranged from 90.4 to 113.6%, and matrix effect varied from 89.3 to 111.0%. All compounds demonstrated stability under relevant conditions. Application of the method to 57 blood samples from 18 patients demonstrated high interpatient variability, with ORE plasma concentrations ranging from 25.6 to 89.9%. The validated LC-MS/MS method provides a feasible, specific, and rapid approach for quantification of BTKis in clinical settings. Simultaneous determination of four BTKis and their metabolites in a single extraction process and chromatographic run reduces analysis time, cost, and resources. The observed variability among individuals highlights the value of TDM for personalized treatment.
布鲁顿酪氨酸激酶抑制剂（BTKis）表现出显著的个体间药代动力学差异，使得治疗药物监测（TDM）成为个性化治疗的一种有前景的方法。然而，同时定量多种BTKis存在技术挑战。临床上需要一种统一的BTKis检测方案。在此，我们开发并验证了一种新型液相色谱-串联质谱（LC-MS/MS）方法，用于同时分析人血浆中的四种BTKis，包括伊布替尼（IBR）、泽布替尼（ZAN）、奥布替尼（ORE）和阿卡替尼（ACB）以及IBR和ACB的活性代谢物（分别为DIH和ACBM）。样品通过使用叔丁基甲基醚的液-液萃取法制备。伊布替尼-d4（IS）用作内标。在XBridge C18柱上进行色谱分离，并连接到与API 4000质谱仪联用的LC-30AD系统。流动相由含0.1%甲酸的10 mM醋酸铵和含0.1%甲酸的乙腈组成。选择优化的m/z 441.4 → 138.3、475.4 → 304.2、472.5 → 455.5、428.3 → 411.5、466.1 → 372.2、482.2 → 388.4和445.5 → 142.5的多反应监测转换分别用于检测IBR、DIH、ZAN、ORE、ACB、ACBM和IS。该方法对所有分析物在1至1000 ng/mL范围内表现出线性（r > 0.99），日内和日间精密度为1.8%至9.7%，准确度低于15%。回收率在90.4%至113.6%之间，基质效应在89.3%至111.0%之间。所有化合物在相关条件下均表现出稳定性。将该方法应用于18例患者的57份血样，显示出患者间的高度变异性，ORE血浆浓度范围为25.6%至89.9%。经过验证的LC-MS/MS方法为临床环境中BTKis的定量提供了一种可行、特异且快速的方法。在单一萃取过程和色谱运行中同时测定四种BTKis及其代谢物可减少分析时间、成本和资源。观察到的个体间变异性突出了TDM在个性化治疗中的价值。
